title: Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes.
abstract: Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, beta-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, M316 , University of Western Australia, Crawley, Western Australia 6009, Australia.
authors.forename: Holger
authors.initials: H
authors.lastname: Eubel
authors.forename: Etienne H
authors.initials: EH
authors.lastname: Meyer
authors.forename: Nicolas L
authors.initials: NL
authors.lastname: Taylor
authors.forename: John D
authors.initials: JD
authors.lastname: Bussell
authors.forename: Nicholas
authors.initials: N
authors.lastname: O'Toole
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Heazlewood
authors.forename: Ian
authors.initials: I
authors.lastname: Castleden
authors.forename: Ian D
authors.initials: ID
authors.lastname: Small
authors.forename: Steven M
authors.initials: SM
authors.lastname: Smith
authors.forename: A Harvey
authors.initials: AH
authors.lastname: Millar
mesh_terms: D017360:Arabidopsis / Q000378:metabolism* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D002458:Cell Fractionation / Q000379:methods; D046508:Culture Techniques; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D020675:Peroxisomes / Q000378:metabolism* / Q000648:ultrastructure; D020543:Proteome; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000032:analysis
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins